Tumor metastasis is a primary cause of death in cancer patients. The overall goal of this research program is to develop key new insights into regulation of tumor metastasis through the study of tumor cell-endothelial cell interaction. The specific goal of this project is to investigate the role of membrane-bound ephrin-A1, a prototypic ligand for EphA receptor tyrosine kinases, in two critical steps of tumor metastasis, vessel recruitment and tumor cell intravasation. Based on our preliminary data and reports from other laboratories, we hypothesize that ephrin-A1 promotes tumor metastasis through coordinated induction of angiogenic factors and direct activation of EphA2 receptors on vascular endothelial cells. To test this hypothesis, Specific Aim 1 will investigate how soluble angiogenic factors, VEGF and leptin, and membrane-bound ephrins coordinate recruitment of blood vessels. We will determine whether ephrin-A1 regulates VEGF and leptin separately or if induction of leptin is prerequisite for elevation of VEGF. The role of leptin in cancer will be determined in vivo by using leptin neutralizing antibodies or soluble leptin receptors. The specific role of leptin in tumor angiogenesis will be analyzed in endothelial-specific leptin receptor knock out mice. Specific Aim 2 will dissect ephrin-A1/EphA2 interaction and signaling in tumor angiogenesis and intravasation. Two novel model systems, the chick embryo metastasis model and the tumor-microenvironment bioreactor, will be used for visualization and quantification of tumor cell intravasation. To study ephrin-A1/EphA2 signaling, an EphA2-null temperature-sensitive immortalized endothelial cell system will be reconstituted with wild-type or mutant EphA2 receptors and tested for binding to interacting proteins, activation of downstream signaling pathways, and the tumor cell-endothelial cell interaction that is required for angiogenesis and intravasation. Specific Aim 3 will determine the role of ephrin-A1 in tumor metastasis in MMTV-Neu and MMTV-PyMT transgenic breast cancer models, using newly created ephrin-A1 knock out mice. Taken together, the success of this project will not only provide critical insights into the rate-limiting steps of tumor metastasis, but also provide new potential therapeutic targets for treatment of this devastating disease. PUBLIC HEALTH RELEVANCE: The goal of this project is to investigate the mechanisms by which cancer cells disseminate into secondary organs in a process called metastasis. Two critical rate-limiting steps in metastasis involve the interaction of tumor cells and blood vessels, in which a novel molecule, termed ephrin-A1, may play a critical role. Experiments proposed in this application to test ephrin-A1 function in tumor metastasis, and success of this project will not only provide critical insights into the rate-limiting steps of tumor metastasis but also provide new potential therapeutic targets to inhibit this devastating disease.